Characterization of thermophilic fungi producing extracellular lignocellulolytic enzymes for lignocellulosic hydrolysis under solid-state fermentation

Abstract

Background

Thermotolerant lignocellulolytic enzymes have become a subject of interest in industrial processes due to their ability to degrade lignocellulosic polysaccharides. Development of cost-effective, large-scale screening for production of desirable enzymes by thermophilic fungi is a challenge. The present investigation focused on isolating, screening, and identifying industrially relevant thermophilic producers of lignocellulolytic enzymes from various locations in the Warangal district, Telangana, India.

Results

Fifteen thermophilic fungi were isolated from soil on their ability to grow at 50 °C and were screened for their activity of cellulase, hemicellulase, and lignin degradation based on holo zone around colonies. The appearance of the black color zone of diffusion in esculin agar is a positive indication for the β-glucosidases activity test. Out of fifteen isolates, Aspergillus fumigatus JCM 10253 have shown as a potential producer of extracellular enzymes for lignocelluloses degradation showing higher activity for cellulase (EI 1.50) as well as β-glucosidase (4 mg/mL), simultaneously for xylanase (EI 1.18) by plate assay methods. A. fumigatus JCM 10253 was selected for extracellular hydrolytic enzymes production under solid-state fermentation. Maximum CMCase (26.2 IU/mL), FPase (18.2 IU/mL), β-glucosidase (0.87 IU/mL), and xylanase (2.6 IU/mL) activities were obtained after incubation time of 144 h at 50 °C. The thermostability of crude cellulase showed the optimum activity at 60 °C and for FPase, β-glucosidase, and xylanase at 50 °C which recommended that the enzymes have a potentially significant role in the biofuel industries.

Conclusion

The high titer production of active enzymes that cleave different β-1,4-glycosidic bonds still remains a challenge and is the major bottleneck for the lignocellulosic conversion. In particular, the finding of thermostable enzymes which would allow the development of more robust processes is a major goal in this field.

Keywords

Lignocellulolytic enzymes / Cellulase / Hemicellulase / β-Glucosidase / Aspergillus fumigatus / Hydrolytic enzymes / Solid-state fermentation

References

[1] Abdel-Sater M, El-Said A. Xylan-decomposing fungi and xylanolytic activity in agricultural and industrial wastes. Int Biodeterior Biodegradation, 2001, 47: 15-21.

[2] Adsul M, Sharma B, Singhania RR, Saini JK, Sharma A, Mathur A, Gupta R, Tuli DK. Blending of cellulolytic enzyme preparations from different fungal sources for improved cellulose hydrolysis by increasing synergism. RSC Adv, 2014, 4: 44726-44732.

[3] Ahirwar S, Soni H, Prajapati BP, Kango N. Isolation and screening of thermophilic and thermotolerant fungi for production of hemicellulases from heated environments. Mycology, 2017, 8(3): 125-134.

[4] Ali A, Muid S. Effect of media and temperature on growth and preliminary detection of ligninolytic and cellulolytic activity of Trametes spp. Borneo J Resour Sci Technol, 2012, 2: 33-40.

[5] Ang S, Shaza E, Adibah Y, Suraini A, Madihah M. Production of cellulases and xylanase by Aspergillus fumigatus SK1 using untreated oil palm trunk through solid state fermentation. Process Biochem, 2013, 48: 1293-1302.

[6] Arora R, Behera S, Sharma NK, Singh R, Yadav YK, Kumar S. Sharma NR, Thakur RC, Sharma M, Parihar L, Kumar G. Biochemical conversion of rice straw (Oryza sativa L.) to bioethanol using thermotolerant isolate K. marxianus NIRE-K3. Proceedings of exploring & basic sciences for next generation frontiers, 2014, New Delhi: Elsevier, 143-146.

[7] Arora R, Behera S, Kumar S. Bioprospecting thermophilic/thermotolerant microbes for production of lignocellulosic ethanol: a future perspective. Renew Sustain Energy Rev, 2015, 51: 699-717.

[8] Bagewadi ZK, Ninnekar HZ. Production, purifiation and characterization of endoglucanase from Aspergillus fumigatus and enzymatic hydrolysis of lignocellulosic waste. Int J Biotechnol Biomed Sci, 2015, 1: 25-32.

[9] Bailey MJ, Biely P, Poutanen K. Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol, 1992, 23: 257-270.

[10] Bairagi S. Isolation, screening and selection of fungal strains for potential cellulase and xylanase production. Int J Pharm Sci Invent, 2016, 5(3): 1-6.

[11] Behera S, Arora R, Kumar S (2013) Bioprospecting the cellulases and xylanases thermozymes for the production of biofuels. In: Paper presented at AICHE annual meeting, San Francisco, 3–8 November, 2013

[12] Behera S, Arora R, Nandhagopal N, Kumar S. Importance of chemical pretreatment for bioconversion of lignocellulosic biomass. Renew Sustain Energy Rev, 2014, 36: 91-106.

[13] Birhanli E, Yesilada O. Increased production of laccase by pellets of Funalia trogii ATCC 200800 and Trametes versicolor ATCC 200801 in repeated-batch mode. Enzyme Microb Technol, 2006, 39: 1286-1293.

[14] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248-254.

[15] Bradner J, Gillings M, Nevalainen K. Qualitative assessment of hydrolytic activities in Antarctic microfungi grown at different temperatures on solid media. World J Microbiol Biotechnol, 1999, 15: 131-132.

[16] de Vries RP, Visser J. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiol Mol Biol Rev, 2001, 65: 497-522.

[17] Edberg SC, Trepeta RW, Kontnick CM, Torres AR. Measurement of active constitutive beta-d-glucosidase (esculinase) in the presence of sodium desoxycholate. J Clin Microbiol, 1985, 21: 363-365.

[18] Florencio C, Couri S, Farinas CS. Correlation between agar plate screening and solid-state fermentation for the prediction of cellulase production by Trichoderma strains. Enzyme Res, 2012

[19] Ford M. Medical microbiology, 2014, Oxford: Oxford University Press.

[20] Fu K, Fu S, Zhan H, Zhou P, Liu M, Liu H. A newly isolated wood-rot fungus for laccase production in submerged cultures. BioResources, 2013, 8: 1385-1397.

[21] Galhaup C, Wagner H, Hinterstoisser B, Haltrich D. Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens. Enzyme Microb Technol, 2002, 30: 529-536.

[22] Gao J, Weng H, Zhu D, Yuan M, Guan F, Xi Y. Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover. Bioresour Technol, 2008, 99: 7623-7629.

[23] Garvey M, Klose H, Fischer R, Lambertz C, Commandeur U. Cellulases for biomass degradation: comparing recombinant cellulase expression platforms. Trends Biotechnol, 2013, 31: 581-593.

[24] Ghose T. Measurement of cellulase activities. Pure Appl Chem, 1987, 59: 257-268.

[25] Grigorevski-Lima A, Da Vinha F, Souza D, Bispo A, Bon E, Coelho R, Nascimento R. Aspergillus fumigatus thermophilic and acidophilic endoglucanases. Appl Biochem Biotechnol, 2009, 155: 18-26.

[26] Grover AK, MacMurchie DD, Cushley RJ. Studies on almond emulsin βd-glucosidase I. Isolation and characterization of a bifunctional isozyme. Biochim Biophys Acta (BBA)-Enzymol, 1977, 482: 98-108.

[27] Hölker U, Höfer M, Lenz J. Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Appl Microbiol Biotechnol, 2004, 64: 175-186.

[28] Hou H, Zhou J, Wang J, Du C, Yan B. Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye. Process Biochem, 2004, 39: 1415-1419.

[29] Ibrahim C. Development of applications of industrial enzymes from Malaysian indigenous microbial sources. Bioresour Technol, 2008, 99: 4572-4582.

[30] Juturu V, Wu JC. Microbial cellulases: engineering, production and applications. Renew Sustain Energy Rev, 2014, 33: 188-203.

[31] Khokhar I, Haider MS, Mushtaq S, Mukhtar I. Isolation and screening of highly cellulolytic filamentous fungi. J Appl Sci Environ Manag, 2012, 16: 223-226.

[32] Kluepfel D. Wood WA, Kellogg ST. Screening of prokaryotes for cellulose-and hemicellulose-degrading enzymes. Methods in enzymology, 1988, Amsterdam: Elsevier, 180-186.

[33] Kovács K, Szakacs G, Zacchi G. Comparative enzymatic hydrolysis of pretreated spruce by supernatants, whole fermentation broths and washed mycelia of Trichoderma reesei and Trichoderma atroviride. Bioresour Technol, 2009, 100: 1350-1357.

[34] Kwon KS, Lee J, Kang HG, Hah YC. Detection of β-glucosidase activity in polyacrylamide gels with esculin as substrate. Appl Environ Microbiol, 1994, 60: 4584-4586.

[35] Lalita, Prasher IB. Qualitative screening of lignocellulolytic enzymes in wood rotting Agaricomycetes from North Western Himalayas. J Adv Bot Zool, 2014

[36] Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev, 2002, 66: 506-577.

[37] Madhavi V, Lele S. Laccase: properties and applications. BioResources, 2009, 4: 1694-1717.

[38] Mallerman J, Papinutti L, Levin L. Characterization of β-glucosidase produced by the white rot fungus Flammulina velutipes. J Microbiol Biotechnol, 2015, 25: 57-65.

[39] Manavalan T, Manavalan A, Heese K. Characterization of lignocellulolytic enzymes from white-rot fungi. Curr Microbiol, 2015, 70: 485-498.

[40] Mandels M, Weber J. The production of cellulases. Adv Chem, 1969, 95: 391-414.

[41] Meddeb-Mouelhi F, Moisan JK, Beauregard M. A comparison of plate assay methods for detecting extracellular cellulase and xylanase activity. Enzyme Microb Technol, 2014, 66: 16-19.

[42] Narasimha Ramulu K, Benarjee G (2016) Diversity and distribution of macrophytes in Nagaram tank of Warangal district, Telangana state

[43] Noratiqah K, Madihah M, Aisyah BS, Eva MS, Suraini A, Kamarulzaman K. Statistical optimization of enzymatic degradation process for oil palm empty fruit bunch (OPEFB) in rotary drum bioreactor using crude cellulase produced from Aspergillus niger EFB1. Biochem Eng J, 2013, 75: 8-20.

[44] Olajuyigbe FM, Nlekerem CM, Ogunyewo OA. Production and characterization of highly thermostable β-glucosidase during the biodegradation of methyl cellulose by Fusarium oxysporum. Biochem Res Int, 2016

[45] Pandey A, Soccol CR, Mitchell D. New developments in solid state fermentation: I-bioprocesses and products. Process Biochem, 2000, 35: 1153-1169.

[46] Pečiulytė D. Isolation of cellulolytic fungi from waste paper gradual recycling materials. Ekologija, 2007, 53(4): 11-18.

[47] Pointing SB. Qualitative methods for the determination of lignocellulolytic enzyme production by tropical fungi. Fungal Divers, 1999, 2: 17-33.

[48] Polizeli M, Rizzatti A, Monti R, Terenzi H, Jorge JA, Amorim D. Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol, 2005, 67: 577-591.

[49] Prasertsan P, H-kittikul A, Chitmanee B. Isolation and selection of cellulolytic fungi from palm oil mill effluent. World J Microbiol Biotechnol, 1992, 8: 614-617.

[50] Raimbault M. General and microbiological aspects of solid substrate fermentation. Electron J Biotechnol, 1998, 1: 26-27.

[51] Ramanjaneyulu G, Reddy GPK, Kumar KD, Reddy BR. Isolation and screening of xylanase producing fungi from forest soils. Int J Curr Microbiol App Sci, 2015, 4: 586-591.

[52] Revankar MS, Lele S. Enhanced production of laccase using a new isolate of white rot fungus WR-1. Process Biochem, 2006, 41: 581-588.

[53] Ruegger MJS, Tauk-Tornisielo SM. Atividade da celulase de fungos isolados do solo da Estação Ecológica de Juréia-Itatins, São Paulo, Brasil. Rev Bras Bot, 2004, 27: 205-211.

[54] Sánchez C. Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnol Adv, 2009, 27: 185-194.

[55] Sandhya C, Sumantha A, Szakacs G, Pandey A. Comparative evaluation of neutral protease production by Aspergillus oryzae in submerged and solid-state fermentation. Process Biochem, 2005, 40: 2689-2694.

[56] Saqib AAN, Whitney PJ. Esculin gel diffusion assay (EGDA): a simple and sensitive method for screening β-glucosidases. Enzyme Microb Technol, 2006, 39: 182-184.

[57] Sherief A, El-Tanash A, Atia N. Cellulase production by Aspergillus fumigatus grown on mixed substrate of rice straw and wheat bran. Res J Microbiol, 2010, 5: 199-211.

[58] Singh S, Dutt D, Tyagi C. Screening of xylanases from indigenously isolated white rot fungal strains for possible application in pulp biobleaching. Open Access Sci Rep, 2013, 2: 262.

[59] Singhania RR, Sukumaran RK, Patel AK, Larroche C, Pandey A. Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for microbial cellulases. Enzyme Microb Technol, 2010, 46(7): 541-549.

[60] Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A. Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresour Technol, 2013, 127: 500-507.

[61] Sohail M, . Distribution of hydrolytic enzymes among native fungi: Aspergillus the pre-dominant genus of hydrolase producer. Pak J Bot, 2009, 41: 2567-2582.

[62] Suwannarangsee S, Bunterngsook B, Arnthong J, Paemanee A, Thamchaipenet A, Eurwilaichitr L, Laosiripojana N, Champreda V. Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design. Bioresour Technol, 2012, 119: 252-261.

[63] Teather RM, Wood PJ. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol, 1982, 43: 777-780.

[64] Tekere M, Zvauya R, Read JS. Ligninolytic enzyme production in selected sub-tropical white rot fungi under different culture conditions. J Basic Microbiol, 2001, 41: 115-129.

[65] Thurston CF. The structure and function of fungal laccases. Microbiology, 1994, 140: 19-26.

[66] Veena V, Poornima P, Parvatham R, Kalaiselvi K. Isolation and characterization of β-glucosidase producing bacteria from different sources. Afr J Biotechnol, 2011, 10: 14891-14906.

[67] Wood PJ, Erfle JD, Teather RM. Wood WA, Kellogg ST. Use of complex formation between Congo Red and polysaccharides in detection and assay of polysaccharide hydrolases. Methods in enzymology, 1988, Amsterdam: Elsevier, 59-74.

[68] Xu X, Lin M, Zang Q, Shi S. Solid state bioconversion of lignocellulosic residues by Inonotus obliquus for production of cellulolytic enzymes and saccharification. Bioresour Technol, 2018, 247: 88-95.

[69] Xue DS, Liang LY, Zheng G, Lin DQ, Zhang QL, Yao SJ. Expression of Piromyces rhizinflata cellulase in marine Aspergillus niger to enhance halostable cellulase activity by adjusting enzyme-composition. Biochem Eng J, 2017, 117: 156-161.

[70] Yadav SK. Technological advances and applications of hydrolytic enzymes for valorization of lignocellulosic biomass. Bioresour Technol, 2017

[71] Yamada R, Hasunuma T, Kondo A. Endowing non-cellulolytic microorganisms with cellulolytic activity aiming for consolidated bioprocessing. Biotechnol Adv, 2013, 31: 754-763.